Project Summary/Abstract: Changes in cancer metabolism have been labeled as a cancer hallmark and many recent reports have focused on the interrogation of metabolism with oncogenic progression. Currently, methods though to assess metabolism are lacking both in sensitivity and throughput. Moreover, measurements of metabolic flux, which govern the flow through a metabolic pathway, are very difficult to acquire on mass limited samples. There remains a critical need to develop an approach that is sensitive (requiring small cell numbers) and high- throughput (assaying multiple cell samples in parallel in short time periods). Methods have recently been developed to increase the signal of nuclear magnetic resonance (NMR) spectroscopy utilizing hyperpolarized molecules. This can afford a dramatic increase in NMR sensitivity of greater than 10,000-fold, but systems needed to take advantage of this for assaying in vitro cell cultures are lacking. We and others have developed in cell NMR methods to assay living cells non-invasively but these require on the order of 108-109 cells, well outside of novel cancer models, such as stem cells and organoids as well as any kind of high-throughput in vitro assay. The objective of this innovative analysis proposal is to develop a micro-coil (Coil) NMR platform which would marry the sensitivity of extremely small volume Coils with the ability to rapidly introduce hyperpolarized molecules and non-invasively monitor metabolic fluxes in less than 104 cells. This enhancement would have a 10,000-fold increased sensitivity over traditional NMR approaches and nearly 1000-fold over destructive LC/MS methods. In Aim 1, we will develop a single-well Coil approach, which can measure metabolic flux on 104 cells and optimize homogeneity with validation as compared to immortal cancer cell lines. In Aim 2, we will convert this single well system in a multi-well and multi-Coil detection scheme, parallelizing the measurement. It is the overarching goal of this proposal to develop a robust benchtop platform, which will transform the approach to metabolic flux analysis in living cells. Utilizing a ?lab on a chip? type approach this platform will be low cost and thus readily able to be disseminated to change the way we characterize cancer biology.